An international group of reference and university hospital laboratories with considerable experience with MRSA and PFGE collaborated to develop a European MRSA strain collection and a PFGE protocol that resulted in a high level of intercenter gel comparability. Strains were contributed from all participating countries, and an internet-accessible European MRSA database was established. Except for the DNA concentration in the plug, standardization of the DNA preparation was not necessary, as each center had already optimized these steps for its own laboratory. However, the group agreed that a standardized protocol for new or inexperienced workers should include details of the desirable cell suspension based on the protocols used in laboratories 1 and 2. One used a standard mold (1 mm thick) and the other used an ultrathin mold (0.5 mm thick), but they produced comparable results.
One problem that was highlighted in our study was the disparity of the temperature measurement in different CHEF apparatuses. The companies are going to reinforce their advice to customers This detail would not have been identified but for an exchange of workers between laboratories, which further illustrates the value of such projects and the detail that is required in methodological descriptions to attain the goal of intercenter standardization and comparability of strains.
The ultimate goal of standardization is to facilitate intercenter comparisons of isolates and strains, aiming at determining their degree of relatedness. In the second phase of the study, four of the seven gels analyzed resulted in almost complete concordance, while three achieved an acceptable level of reproducibility. This highlights the need for laboratories to reevaluate their protocols if consistently suboptimal results are obtained, for example, due to plugs overloaded with DNA or blocks that have not been stored appropriately.
The second phase of the study also showed that many, but clearly not all, of the methodological problems seen in phase I were eventually overcome by close cooperation between laboratories and standardization of certain parameters. The most important variable appeared to be the skill of the operator, as overloading of the gel and inadequate destaining were evident. We therefore recommend including an internal quality control to assess the reproducibility and quality of the gel before inclusion in the database for intra- or intercenter comparison. The quality control strain should ideally demonstrate >95% similarity between gels to ensure acceptability. When the gels from laboratories 1 and 2 were compared by using GelCompar, almost 100% correlation was observed between corresponding isolates from each. There were a few minor differences observed, particularly with respect to the strains that make up the major clonal group called cluster A in this study. This group, otherwise known as the Iberian clone, was confirmed by comparison with the strain identified as being the prototype submitted to the HARMONY collection by Herminia de Lencastre (22
). It emerged in the early 1990s and had been previously characterized by other techniques (1
A recent multicenter study by Chung et al. (9
) compared gels run in different laboratories by using a fully standardized protocol. It was unclear how much experience with the PFGE technique these laboratories had before participating in this study. They showed that, even when the full process was standardized, some of the problems (amount of loaded DNA; overstaining or under-destaining of the gel; too much DNA in the blocks, indicating insufficient lysis; blocks not stored properly; and problems related to the electrophoresis) experienced in both phases of our study also occurred in that study. Again, this highlights the importance of using skilled operators for performing the technique if we are to aspire to national and international comparison and tracking of strains.
The Nordic countries (Denmark, Finland, Norway, and Sweden) have produced a standardized protocol that is used for interlaboratory comparison (C. S. Elsberg, S. Salmenlinna, J. V. Fussing, J. Vuopio-Varkila, V. T. Rosdahl, and the Nordic MRSA Study Group. Abstr. 9th Int. Symp. Staphylococci Staphylococcal Infect., abstr. 90, 2000). The protocol was based on the most common parameters from the in-house PFGE protocols in 8 of the 12 laboratories involved. Overall, there was very good correlation between the laboratories, confirmed by visual and BioNumerics (Applied Maths) analysis. They found that interlaboratory comparison of MRSA could be achieved, as the minor differences observed could be attributed to the poor resolution of TIFF files exchanged. In particular, they found that attention should be paid to the variable photographic quality in the lower part of the gels (between 48.5 and 97kb, when lambda is used as a reference standard). They also highlighted the need for a reduction in the workload and identification time.
Standardization of the image capture process and optimal settings for analysis are also required. In this study, it was necessary to standardize the size of TIFF files to allow comparison by using GelCompar or BioNumerics analysis software. For intercenter comparisons, it is important to specify the part of the gel that is to be included in the analysis. Bands above the highest-molecular-size band of the NCTC 8325 marker (674 kb) should not be included, as this part of the gel will not have been normalized. Similarly, bands below the third-lowest band of the marker (36 kb) should also be excluded due to the often-poor resolution of the lower-molecular-size bands.
The exchange of isolates and central analysis revealed the international spread of MRSA clones in the EU. This proved to be far more extensive than had been realized previously and justified the establishment of the HARMONY network, which the IUMS subcommittee now wishes to extend globally. Already several other countries have joined the network, and additional techniques have been performed to further explore the clonality of the EMRSA strains (unpublished data).
Following from a previous study by Deplano et al. (13
), an extremely important outcome of this study is that a number of clonally related strains have been shown to be present in multiple countries throughout Europe. The well-known Iberian clone (PFGE cluster A) has been demonstrated in Belgium, Finland, France, Germany, and Spain (and from the wider HARMONY collection in Portugal, Slovenia, and Sweden). Other strains in the collection (from Greece, the United Kingdom, and Ireland) may also be related, although more distantly, which supports the clonal nature of MRSA (2
). Other clonally related strains have also been identified. For example, the South German II strain is closely related to the most prevalent strain in Slovenia. The United Kingdom strains EMRSA-15 and EMRSA-16 (which belongs to PFGE cluster B) have recently been identified in a number of other European countries as a result of this study. EMRSA-15 has been found in Finland (S. Salmenlinna, personal communication), Germany (W. Witte, personal communication), and Sweden, while EMRSA-16 has been identified in Finland via Turkey, in Sweden via Cyprus, and Denmark via Sweden (C. S. Elsberg, J. Mondrup, L. Larsson, S. Murchan, and C. Welinder-Olsson, Abstr. 9th Int. Symp. Staphylococci Staphylococcal Infect., abstr. 232, 2000). Studies have also shown that this strain is present in Switzerland (6
), and this highlights the need for closer international collaboration to monitor the spread of current epidemic strains as well as the emergence of new ones. Furthermore both EMRSA-15 and EMRSA-16 have been identified in Belgium since at least 2001 in six and two hospitals, respectively (M. Struelens and coworkers, personal communication). PFGE cluster C, representing strains in Belgium and Finland, may also be distantly related to the South German II and United Kingdom EMRSA-3 strains.
Reliable and reproducible intercenter comparisons are possible by using the standardization approach outlined here (an approach we have termed harmonization). However, as with many other techniques, it is important to remember that the largest impediment to getting reproducible results is the skill of the operator. At least 5 of the 10 laboratories have adopted this new protocol for routine use in epidemiological studies, and such an approach will be required if laboratories are to externally query the IUMS HARMONY database that is soon to be established by using the BioNumerics software package in the coordinating laboratory. Other laboratories have also agreed to use this new protocol for the purpose of comparing strains with the central database. This internet-based database with the reference profiles (type strains and predominant subtypes) will be maintained by the coordinating laboratory as the IUMS reference center and administered via the IUMS subcommittee. Other laboratories performing PFGE will be able to compare their isolates of interest with the reference collection. These isolates should then be submitted to the central laboratory for validation once the agreed national center has confirmed that it is a new strain by repeating the PFGE with the relevant reference strain by both computer-assisted and visual comparison. It will then be added to the collection and reference panel library.
The database includes antimicrobial susceptibility and toxin typing characteristics, and more will be added, including other genotypic data, in the near future. Some of these characteristics are helpful in interpreting the relationship and evolution of strains within a country (12
). However, interpreting them can be quite a challenge: some toxins, for example, are known to be phage related, and perhaps the variations we have observed may be related to this process (10
The group was aware of the PulseNet food-borne enteric disease surveillance network that had been established in four laboratories in 1995 (25
). PulseNet had shown that when a protocol was strictly followed, near-perfect intercenter reproducibility was possible for most of the participating laboratories. There is now a rigorous quality assurance and accreditation system in place. Interestingly, when a laboratory introduced an unspecified variation in the protocol, a problem with interlaboratory comparison occurred. It was not deemed appropriate to adopt this approach ab initio with the HARMONY laboratories. Instead, we wanted to learn from each other's expertise and experience. The final protocol is thus “owned” by the group and seen to be “fit for the purpose.” It has been adopted by all of the laboratories for interlaboratory comparisons and by several for all their routine work. It will be interesting to see if, as experience and further quality assurance work progress, all laboratories are willing to use the protocol for their routine work and build new databases for their own countries.
A similar Canadian PFGE comparison and standardization study was published (19
) just as the work of HARMONY was completed. Their results look very promising, although they are working further to improve their intercenter reproducibility, using, at this juncture, five quality assurance strains. It will be important for the IUMS to continue progression of the HARMONY initiative globally, discuss the various published and other initiatives, and build on the whole group's experiences and opinions.
We are also examining other typing methods, such as use of methicillin resistance genes (mecRIA and SCCmec), multilocus sequence typing, binary typing, and ribotyping, to establish which of these will be recommended and when, to confirm the relationships between known PFGE patterns, and to confirm the relationships of new strains to those already in the collection. There is still a need to address the international nomenclature of strains and the relative roles and utility of the various typing systems in this process. It is the intention of the IUMS subcommittee to work on this. The results of epidemiological typing data on all MRSA isolates from bloodstream infections (in collaboration with the European Antimicrobial Resistance Surveillance System) and other clinical and carriage isolates could give further information on the spread of EMRSA clones throughout Europe.
In conclusion, the HARMONY typing group has established a European collection and database of EMRSA strains and developed a harmonized protocol for PFGE that has been widely adopted for international comparisons, thus providing important information on spread of epidemic clones of MRSA.